Hydrolytic enzymes involved in rotifer digestive processes were investigated directly at the sites of enzyme action using the ELF (Enzyme Labelled Fluorescence) technique. After enzymatic hydrolysis of an artificial ELF substrate, the fluorescent product ELF alcohol (ELFA) marked the sites of enzyme action. The time development of ELFA labelling was studied at different incubation times. Phosphatases, beta-N-acetylhexosaminidases and lipases were examined in Brachionus angularis, B. calyciflorus, Keratella cochlearis and Lecane closterocerca from fed-batch cultures. We detected activities of all studied enzymes mostly in the stomach and intestine of rotifers. L. closterocerca was the only species showing enzyme activity at the mastax. Lipase activity was observed in the stomach and intestine of all species and in the mastax of L. closterocerca. Phosphatases were frequently located at the corona of B. calyciflorus. In other cases, both phosphatases and beta-N-acetylhexosaminidases were rarely detected at the corona, and on the lorica and epidermis of some species.